Multi-angle light scattering (MALS) is often used to characterize molar mass and size of macromolecules in solution by measuring the light scattering properties of the solution as it passes through a flow cell. MALS may also be used to characterize macromolecular interactions in solution by measuring the light scattering properties from a series of sample concentrations or compositions which are injected into the flow cell, usually with a stopped-flow period following the injection. The analysis of MALS data to determine molar mass and interaction properties generally requires knowledge of the concentration of the macromolecule in the MALS cell. The concentration of molecules in solution may be determined by measurement of the sample solution with a concentration detector such as a ultra-violet/visible light absorption (UV/Vis) spectrophotometer or differential refractometer. Typically the concentration detector comprises a flow cell separate from the MALS cell, often contained in an entirely different enclosure or instrument, the two cells being connected by capillary tubing so that sample passes from one cell to the other sequentially. Alternatively, the measurement may be made simultaneously by both instruments by splitting the flow between them with the correct split flow ratio.
Simultaneous, split-flow measurement may introduce certain systematic errors in data interpretation since the concentration in the MALS cell may not be identical to that in the concentration detector cell. This can be due, in part, to inaccuracy of the initial split flow ratio between the two cells, as well as the flow ratio changing in the course of a measurement due to changes in solution viscosity or in capillary dimensions as a result of thermal expansion or clogging. The systematic errors may be partially overcome by increasing the volume of sample delivered to the detectors, but this is undesirable as many samples of interest are not available in sufficient quantity.
In the case of sequential flow, an error in concentration measurement may occur due to the finite mixing volumes of the cells and intermediate capillary tubing, often referred to as band broadening. As each fraction passes through a detection device, it produces a signal often referred to as a “band” or “peak.” Because of dispersion and mixing effects, these bands are broadened somewhat each time the sample passes through a different device. Consider a sample comprised of a low concentration aliquot of a monodisperse protein. In this event, the excess Rayleigh ratio is directly proportional to the molar mass and the concentration. The light scattering signal and the concentration signal should be of identical shape and would overlay perfectly were the two responses normalized to have the same areas. However, as the sample passes from the MALS detector and enters the concentration detector, it passes through intermediate regions and connections that contribute to the dispersion and mixing of the sample. Band broadening and mathematical methods for correcting its effects are described in U.S. Pat. No. 7,386,427, “Method for correcting the effects of interdetector band broadening,” issued on Jun. 10, 2008, by S. Trainoff, and U.S. Pat. No. 7,911,594, “Method to derive physical properties of a sample after correcting the effects of interdetector band broadening,” issued on Mar. 22, 2011, also by S. Trainoff, both of which are incorporated herein by reference.
The structure of the flow cells can add significantly to the measurement capabilities of the respective instruments. For example, the MALS cell discussed in U.S. Pat. No. 4,616,927, “Sample cell for light scattering measurements,” by Steven D. Phillips, et. al. issued Oct. 14, 1986, and U.S. Pat. No. 4,907,884, “Sample cell monitoring system,” by P. Wyatt et. al., issued Mar. 13, 1990, both patents herein incorporated by reference, disclose the use of a cylindrical flow cell through which has been drilled a bore. The sample passes through the bore, the cylindrical cell acts as a lateral lens, and the liquid glass interfaces are far removed from the scattering volume, thereby enabling the examination of the light scattered by the solution at virtually all scattering angles without introducing significant background artifacts from the cell itself.
The sensitivity of measurements made within a UV/Vis cell is greatly affected by the path length being traversed by the UV light source. The Beer-Lambert Law states:A=εcl  (1)where A is the absorbance, ε is the molar absorption coefficient, c is the concentration of the sample and l is the path length traversed by the UV beam through the sample. If, then, A is measured by the spectrophotometer, and the ε and l are known, the concentration c may be determined. Further, the longer the path length, l, the greater the sensitivity of the measurement of the concentration is likely to be.
It is an objective of the present invention to minimize band broadening between MALS and UV measurements while improving the overall sensitivity of the concentration detection by enabling a variable UV beam path length. It is a further objective of the invention to facilitate the integration of both UV/Vis absorption and MALS detection within a single analytical instrument as well as to enable such a dual measurement system, via a drop-in/modular modification, into an existing MALS-only detection instrument without need for extensive instrument alteration or re-design.